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US010208322B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 ,208 , 322 B2 Coelho et al. (45 ) Date of Patent: * Feb . 19, 2019 ( 54 ) IN VIVO AND IN VITRO OLEFIN ( 56 ) References Cited CYCLOPROPANATION CATALYZED BY HEME ENZYMES U . S . PATENT DOCUMENTS 3 , 965 ,204 A 6 / 1976 Lukas et al. (71 ) Applicant: California Institute of Technology , 4 , 243 ,819 A 1 / 1981 Henrick Pasadena , CA (US ) 5 ,296 , 595 A 3 / 1994 Doyle 5 , 703 , 246 A 12 / 1997 Aggarwal et al. 7 , 226 , 768 B2 6 / 2007 Farinas et al. ( 72 ) Inventors : Pedro S . Coelho , Los Angeles, CA 7 , 267 , 949 B2 9 / 2007 Richards et al . (US ) ; Eric M . Brustad , Durham , NC 7 ,625 ,642 B2 12 / 2009 Matsutani et al. (US ) ; Frances H . Arnold , La Canada , 7 ,662 , 969 B2 2 / 2010 Doyle et al. CA (US ) ; Zhan Wang , San Jose , CA 7 ,863 ,030 B2 1 / 2011 Arnold (US ) ; Jared C . Lewis , Chicago , IL 8 ,247 ,430 B2 8 / 2012 Yuan 8 , 993 , 262 B2 * 3 / 2015 Coelho . .. .. .. • * • C12P 7 /62 (US ) 435 / 119 9 ,399 , 762 B26 / 2016 Farwell et al . (73 ) Assignee : California Institute of Technology , 9 , 493 ,799 B2 * 11 /2016 Coelho .. C12P 7162 Pasadena , CA (US ) 2006 / 0030718 AL 2 / 2006 Zhang et al. 2006 / 0111347 A1 5 / 2006 Askew , Jr . et al. 2007 /0276013 AL 11 /2007 Ebbinghaus et al . ( * ) Notice : Subject to any disclaimer , the term of this 2009 /0238790 A2 9 /2009 Sommadosi et al. patent is extended or adjusted under 35 2010 / 0056806 A1 3 / 2010 Warren U . S . C . 154 (b ) by 0 days . 2010 /0168463 A1 7 /2010 Hirata et al. This patent is subject to a terminal dis 2010 /0240106 AL 9 /2010 Wong et al . claimer . 2011/ 0196086 A18 / 2011 Matsushita et al. ( Continued ) (21 ) Appl. No. : 15 / 278 , 561 FOREIGN PATENT DOCUMENTS (22 ) Filed : Sep . 28, 2016 EP 0 200 638 B1 4 / 1986 (65 ) Prior Publication Data WO 2007 / 144599 A2 12 / 2007 US 2017 / 0247725 A1 Aug. 31 , 2017 (Continued ) OTHER PUBLICATIONS Related U . S . Application Data Adams, P . et al ., “ Phenix : a comprehensive Python -based system for (63 ) Continuation of application No . 14 /625 , 449, filed on macromolecular structure solution ,” Acta Crystallogr ., Sect . D , Feb . 18 , 2015 , now Pat . No . 9 ,493 ,799 , which is a Biol. Crystallogr ., 2010 , D66 ( 2 ) : 213 -221 . continuation of application No . 14 / 185 , 861 , filed on ( Continued ) Feb . 20 , 2014 , now Pat . No . 8 , 993 , 262 , which is a continuation of application No . PCT /US2013 / 063577 , filed on Oct . 4 , 2013 . Primary Examiner — Pancham Bakshi (74 ) Attorney, Agent, or Firm — Kilpatrick Townsend & (60 ) Provisional application No . 61/ 711 ,640 , filed on Oct. Stockton LLP 9 , 2012 , provisional application No . 61 /740 , 247 , filed on Dec . 20 , 2012 , provisional application No . ABSTRACT 61 /784 ,917 , filed on Mar . 14 , 2013 , provisional (57 ) application No . 61/ 838 , 167 , filed on Jun . 21, 2013 , The present invention provides methods for catalyzing the provisional application No. 61/ 815 ,997 , filed on Apr . conversion of an olefin to any compound containing one or 25 , 2013 , provisional application No . 61/ 818 , 329 , more cyclopropane functional groups using heme enzymes . filed on May 1 , 2013 , provisional application No . In certain aspects , the present invention provides a method 61/ 856 ,493 , filed on Jul. 19 , 2013 . for producing a cyclopropanation product comprising pro (51 ) Int. CI. viding an olefinic substrate , a diazo reagent, and a heme C12P 13 / 02 ( 2006 .01 ) enzyme; and admixing the components in a reaction for a C12P 7762 ( 2006 . 01 ) time sufficient to produce a cyclopropanation product. In C12N 9 /02 ( 2006 . 01 ) other aspects , the present invention provides heme enzymes (52 ) U . S . CI. including variants and fragments thereof that are capable of CPC . .. .. C12P 13 /02 (2013 . 01 ) ; C12N 9 / 0004 carrying out in vivo and in vitro olefin cyclopropanation (2013 .01 ) ; C12N 9 /0042 (2013 . 01 ) ; C12P 7762 reactions . Expression vectors and host cells expressing the ( 2013 . 01 ) ; C12Y 106 / 02004 ( 2013 .01 ) ; YO2P heme enzymes are also provided by the present invention . 20 /52 (2015 . 11 ) (58 ) Field of Classification Search CPC .. .. C12P 13 /02 , C12P 7 /62 ; C12N 9 /0042 7 Claims, 46 Drawing Sheetsts See application file for complete search history . Specification includes a Sequence Listing . US 10 , 208 , 322 B2 Page 2 References Cited Davies , H . and Venkataramani, C . , “ Dirhodium tetraprolinate ( 56 ) catalyzed asymmetric cyclopropanations with high turnover num U . S . PATENT DOCUMENTS bers , ” Org . Lett ., 2003 , 5 ( 9 ) : 1403 - 1406 . 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WO 2013 / 124280 AL 8 / 2013 Doyle , M . et al. , “ Dirhodium ( II ) tetrakis [methyl 2 - oxaazetidine - 4 WO 2013 / 151690 A1 10 / 2013 carboxylate ] : a chiral dirhodium ( II ) carboxamidate of exceptional WO 2014 /058729 A14 / 2014 reactivity and selectivity , " Org . Lett ., 2000 , 2 ( 8 ) : 1145 - 7 . WO 2014 /058744 A2 4 / 2014 Dunford , A . et al ., “ Probing the molecular determinants of coen WO 2016 /086015 A1 6 / 2016 zyme selectivity in the P450 BM3 FADNADPH domain , " Biochimica WO 2016 / 191612 A2 12 / 2016 Biophysica Acta , 2009 , 1794 ( 8 ) : 1181 - 1189 . Emsley, P . and Cowtan , K ., “ Coot : model -building tools for molecu lar graphics, ” Acta Crystallogr ., Sect. D , Biol. Crystallogr. , 2004 , OTHER PUBLICATIONS D60 ( 12 , Pt. 1 ) :2126 -2132 . Ajikumar , P . et al ., “ Isoprenoid pathway optimization for taxol Evans, D . et al. , “ Bis ( oxazolines) as chiral ligands in metal catalyzed asymmetric reactions . Catalytic , asymmetric cyclopropana precursor overproduction in Escherichia coli, ” Science, 2010 , 330 : 70 tion of olefins , " J . Am . Chem . Soc . , 1991, 113 ( 2 ) : 726 - 8 . 74 . Evans, P . , “ Scaling and assessment of data quality, ” Acta Crystal Alliot, J. et al. , “ Enantioselective synthesis of levomilnacipran ," logr . , Sect . D , Biol. Crystallogr. , 2006 , D62( 1 ) :72 - 82 . Chem . Commun . , 2012 , 48 (65 ) :8111 - 8113 . Galardon , E . et al. , “ Insertion of ethyl diazoacetate into N - H and Altschul, S . et al. , “ Basic local alignment search tool, ” J . Mol. Biol ., S - H bonds catalyzed by ruthenium porphyrin complexes, ” J . Chem . 1990 , 215 ( 3 ) :403 - 10 . Perkin Tans, 1 : 2455 - 2456 , 1997 . Bailey, S . , “ The CCP4 suite : programsfor protein crystallography, " Girvan et al. , " Glutamate -Heme Ester Bond Formation Is Disfavoured Acta Cryst . , Sect. D Biol. Crystallogr ., 1994 , D50 ( 5 ) :760 - 763. in Flavocytochrome P450 BM3, ” Biochemical Journal, 2010 , pp . Baumann , L . et al. , “ Iron porphyrin catalyzed N - H insertion reac 455 -466 . tions with ethyl diazoacetate , " Organometallics , 2007 , 26 :3995 Green , M . , “ C - H bond activation in heme proteins : the role of 4002 . thiolate ligation in cytochrome P 450 , ” Curr . Opin . Chem . Biol . , Bergman , R ., “ Organometallic chemistry : C - H activation , ” Nature , 2009 , 13 ( 1 ) : 84 - 88 . 2007 , 446 (7134 ) :391 - 393 . Groves , J . , “ The bioinorganic chemistry of iron in oxygenases and Bloom , J. et al. , " Protein stability promotes evolvability , ” Proc . supramolecular assemblies , ” Proc. Natl . Acad . Sci . U . S . A . , 2003 , 100 ( 7 ) : 3569 - 3574 . Natl. Acad . Sci. U . S . A ., 2006 , 103 ( 15 ) : 5869 - 5874 . Haines , D . et al. , “ Pivotal Role of Water in the Mechanism of Bonnaud , B . et al. , “ 1 - Aryl - 2 - aminomethyl) cyclopropanecarboxylic P450BM - 3 , ” Biochemistry , 2001 , 40 ( 45 ) : 13456 - 13465 . acid derivatives. A new series of potential antidepressants, " J . Med . Heel et al . , “ Non - natural Olefin Cyclopropanation Catalyzed by Chem ., 1987 , 30 ( 2 ) : 318 - 25 . Diverse Cytochrome P450s and Other Hemoproteins” , ChemBioChem Bornscheuer , U . and Kazlauskas , R ., “ Reaction specificity of enzymes : vol. 15 , Iss . 17 , Oct . 7 , 2014 , pp . 2556 - 2562 . Catalytic promiscuity in biocatalysis : Using old enzymes to form Hiraga , K . and Arnold , F ., “General method for sequence new bonds and follow new pathways, ” Angew . Chem . , Int. Ed . , independent site - directed chimeragenesis , " J . Mol . Biol. , 2003 , 2004 , 43 (45 ) :6032 -6040 . 330 ( 2 ) : 287 - 96 . Boyce , M . and Bertozzi , C ., “ Bringing chemistry to life ,” Nat . 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  • Progress in the Production of Medicinally Important Secondary

    Progress in the Production of Medicinally Important Secondary

    Progress in the production of medicinally important secondary metabolites in recombinant microorganisms or plants-Progress in alkaloid biosynthesis Michael Wink, Holger Schäfer To cite this version: Michael Wink, Holger Schäfer. Progress in the production of medicinally important secondary metabo- lites in recombinant microorganisms or plants-Progress in alkaloid biosynthesis. Biotechnology Jour- nal, Wiley-VCH Verlag, 2009, 4 (12), pp.1684. 10.1002/biot.200900229. hal-00540529 HAL Id: hal-00540529 https://hal.archives-ouvertes.fr/hal-00540529 Submitted on 27 Nov 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biotechnology Journal Progress in the production of medicinally important secondary metabolites in recombinant microorganisms or plants-Progress in alkaloid biosynthesis For Peer Review Journal: Biotechnology Journal Manuscript ID: biot.200900229.R1 Wiley - Manuscript type: Review Date Submitted by the 28-Oct-2009 Author: Complete List of Authors: Wink, Michael; Heidelberg University, Institute for Pharmacy and Molecular Biotechnology
  • Molecular Characterization, Protein–Protein Interaction Network, and Evolution of Four Glutathione Peroxidases from Tetrahymena Thermophila

    Molecular Characterization, Protein–Protein Interaction Network, and Evolution of Four Glutathione Peroxidases from Tetrahymena Thermophila

    antioxidants Article Molecular Characterization, Protein–Protein Interaction Network, and Evolution of Four Glutathione Peroxidases from Tetrahymena thermophila Diana Ferro 1,2, Rigers Bakiu 3 , Sandra Pucciarelli 4, Cristina Miceli 4 , Adriana Vallesi 4 , Paola Irato 5 and Gianfranco Santovito 5,* 1 BIO5 Institute, University of Arizona, Tucson, AZ 85719, USA; [email protected] 2 Department of Pediatrics, Children’s Mercy Hospital and Clinics, Kansas City, MO 64108, USA 3 Department of Aquaculture and Fisheries, Agricultural University of Tirana, 1000 Tiranë, Albania; [email protected] 4 School of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy; [email protected] (S.P.); [email protected] (C.M.); [email protected] (A.V.) 5 Department of Biology, University of Padova, 35131 Padova, Italy; [email protected] * Correspondence: [email protected] Received: 6 September 2020; Accepted: 1 October 2020; Published: 2 October 2020 Abstract: Glutathione peroxidases (GPxs) form a broad family of antioxidant proteins essential for maintaining redox homeostasis in eukaryotic cells. In this study, we used an integrative approach that combines bioinformatics, molecular biology, and biochemistry to investigate the role of GPxs in reactive oxygen species detoxification in the unicellular eukaryotic model organism Tetrahymena thermophila. Both phylogenetic and mechanistic empirical model analyses provided indications about the evolutionary relationships among the GPXs of Tetrahymena and the orthologous enzymes of phylogenetically related species. In-silico gene characterization and text mining were used to predict the functional relationships between GPxs and other physiologically-relevant processes. The GPx genes contain conserved transcriptional regulatory elements in the promoter region, which suggest that transcription is under tight control of specialized signaling pathways.